Metabolism of Oat Leaves during Senescence : VII. The Interaction of Carbon Dioxide and Other Atmospheric Gases with Light in Controlling Chlorophyll Loss and Senescence

In air largely freed from CO₂, senescence of isolated oat (Avena sativa cv Victory) seedling leaves is no longer prevented by white light; instead, the leaves lose both chlorophyll and protein as rapidly as in the dark. Senescence in light is also accelerated in pure O₂, but it is greatly delayed in N₂; 100% N₂ preserves both protein and chlorophyll in light and in darkness. In light in air, most of the compounds tested that had previously been found to delay or inhibit senescence in darkness actually promote the loss of chlorophyll, but they do not promote proteolysis. Under these conditions, proteolysis can therefore be separated from chlorophyll loss. But in light minus CO₂, where chlorophyll loss is rapid in controls, two of these same reagents prevent the chlorophyll loss. Unlike the many reagents whose action in light is thus the opposite of that in darkness, abscisic acid, which promotes chlorophyll loss in the dark, also promotes it in light with or without CO₂. Kinetin, which prevents chlorophyll loss in the dark, also prevents it in light minus CO₂. In general, therefore, the responses to light minus CO₂ are similar to the responses to darkness, and (with the exception of abscisic acid and kinetin) opposite to the response to light in air.     

CYCLING OF STOMATAL APERTURE IN LEAVES OF PLANTS WITH CRASSULACEAN ACID METABOLISM UNDER CONSTANT CONDITIONS

When leaves of plants with C₃ metabolism are detached and held in darkness, they senesce and the stomata close. Because the relation of senescence and stomatal closure is very close, if not actually causal, the question arose as to whether in the leaves of plants with Crassulacean acid metabolism whose stomata open at night the relationship to senescence would be reversed. Detached leaves of four species of Hoya, floated on water in constant darkness or constant light, were found to show no large differences in stomatal aperture (measured as diffusion resistance) between those in the light or dark, but the aperture changed in a regular circadian rhythm. In some leaves the rhythm was simple, in others the peak showed small secondary peaks, but in all cases the values were nearly the same in the light as in the dark, throughout the cycle. Previous culture of the intact plants under normal day/night conditions gave results similar to those with plants that had had prolonged culture under constant light or darkness. In those cases when the stomata were more open in the dark, the chlorophyll content was greater than when the stomata were more open in the light; but when they were more open in the light, the chlorophyll content showed little difference between light and dark. When the leaves had only their petioles in water they showed greater senescence in the light than in the dark, and the stomata were more tightly closed in the light, especially at the apical ends. All four species of Hoya gave similar results. We deduce that senescence of these leaves is modified by stomatal aperture, and generally in the same direction as in C₃ leaves, but that in continuous light or darkness the primary control over the aperture is the endogenous cycle.     

Metabolism of Oat Leaves during Senescence: V. Senescence in Light

A comparison has been made of the progress of senescence in the first leaf of 7-day-old oat plants (Avena sativa cv. Victory) in darkness and in white light. Light delays the senescence, and intensities not over 100 to 200 ft-c (1000-2000 lux) suffice for the maximum effect. In such intensities, chlorophyll loss and amino acid liberation still go on in detached leaves at one-third to one-half the rate observed in darkness; however, when the leaves are attached to the plant, the loss of chlorophyll in 5 days is barely detectable. Transfer of the leaves from 1 or 2 days in the low intensity light to darkness, or vice versa, shows no carryover of the effects of the preceding exposure, so that such treatment affords no evidence for the photoproduction of a stable substance, such as cytokinin, inhibiting senescence. Light causes a large increase in invertaselabile sugar and a smaller increase in glucose, and application of 100 to 300 mm glucose or sucrose in the dark maintains the chlorophyll, at least partially. Correspondingly, short exposure to high light intensity, which increased the sugar content, had a moderate effect in maintaining the chlorophyll. However, 3-(3,4-dichlorphenyl)-1,1-dimethylurea (DCMU) completely prevents the increases in sugars and yet does not prevent the effect of light on senescence, whether determined by chlorophyll loss or by protein hydrolysis. Light causes a 300% increase in the respiration of detached oat leaves, and kinetin lowers that only partly, but unlike the increased respiration associated with senescence in the dark, the increase in the light is fully sensitive to dinitrophenol, and therefore cannot be ascribed to respiratory uncoupling. The increased respiration in light is prevented by DCMU, parallel with the prevention of sugar formation. It is therefore ascribed to the accumulation of soluble sugars, acting as respirable substrate. Also, l-serine does not antagonize the light effect. For all of these reasons, it is concluded that the action of light is not mediated by photosynthetic sugar formation, nor by photoproduction of a cytokinin. Instead, we propose that light exerts its effect by photoproduction of ATP. The action of sugars is ascribed to the same mechanism but by way of respiratory ATP. This hypothesis unifies most of the observed phenomena of the senescence process in oat leaves, and helps to explain some of the divergent findings of earlier workers.     

The senescence of detached leaves of tropaeolum

The senescence of detached Tropaeolum majus leaves was compared with that described earlier for Avena. Tropaeolum was chosen as being not only a dicot but also as having a nearly circular leaf, thus needing only the smallest minimum of wounding, since wounding delays the loss of chlorophyll and protein in darkness. Tropaeolum resembles Avena in that closing the stomata osmotically or with ABA causes rapid senescence in light. As in Avena also, n-hexanol and α,α′-dipyridyl delay senescence in darkness but cause `bleaching' of chlorophyll in light. Unlike Avena, however, kinetin and gibberellic acid, which delay senescence in the dark in both species, do so in Tropaeolum without causing any significant stomatal opening. The senescence of Tropaeolum leaves is actually promoted by fusicoccin, which powerfully delays senescence in Avena, although fusicoccin does cause stomatal opening in darkness in both species. Thus, many of the phenomena of senescence are alike in the monocot and dicot, but there are several significantly different responses to the senescence-modifying reagents. It is concluded that while stomatal closure accelerates senescence in both species, stomatal opening is not directly linked to the prevention of leaf senescence.     

The Role of Abscisic Acid in Senescence of Detached Tobacco Leaves

The role of abscisic acid in the regulation of senescence was investigated in detached tobacco leaves (Nicotiana rustica L.). Leaves senesced in darkness showed a sharp rise in abscisic acid level in the early stage of aging, followed by a rapid decline later. The same trend was found when leaves were aged in light, but the rise in abscisic acid occurred four days later than in darkness. Senescence was slower in light than in darkness, while salt stress accelerated the processes. Leaves treated with kinetin which senesced in light and darkness, did not show an increase in abscisic acid. Application of kinetin led to a transformation from free to bound ABA. These results may indicate that ABA and cytokinin are involved in a trigger mechanism which regulates senescence; the stage at which this trigger is activated determines the rate of senescence.     

Influence of Different Cytokinins on the Transpiration and Senescence of Excised Oat Leaves

In order to investigate the possibility that cytokinins control transpiration indirectly through affecting leaf senescence, a direct comparison was made of the effect of different cytokinins on transpiration and senescence of oat leaves (Avena sativa L. cv. Forward). Senescence was assessed by measuring chlorophyll loss. The synthetic cytokinins N⁶ benzyladenine (BA) and kinetin delayed senescence and increased transpiration of oat leaves to a greater extent than did the naturally occurring compounds zeatin, N^b-Δ² isopentenyladenine (i⁶ Ade) and 6-ø-hydroxybenzyladenosine (hyd-BA riboside). During the early stages of the transpiration experiment zeatin showed similar or greater activity than BA. This period was longest when freshly excised leaves were used, was reduced when leaves were used after incubation in distilled water in the dark for 20 h and was eliminated by incubation in cytokinin solution in the dark. After this period the activity of zeatin declined relative to BA. The effect of cytokinins in increasing transpiration occurred only in the light; no effect was observed in the dark. BA showed higher activity than zeatin in senescence tests but both cytokinins were less effective as the tests progressed, this decrease in activity being more rapid when older leaves were used. The results are discussed in relation to the mechanisms by which endogenous cytokinins might control sensecence and transpiration in oat leaves and to the value of the oat leaf senscence and transpiration bioassays as tests for cytokinin activity of plant extracts.     

The relation between nitrogen deficiency and leaf senescence

Because the "mobilization" of nitrogen resulting from nutritional nitrogen deficiency is also prominent during leaf senescence, the characteristics of these two syndromes were compared. Oat plants ( Avena sativa L. cv. Victory) were raised on a nutrient solution, complete except for nitrogen supply (i.e., with only the seed protein as nitrogen source), and the senescence of their leaves was compared with that of controls grown on a full nutrient solution. The N-deficient plants flowered after forming only 4 leaves and each set a single seed. The nitrogen lack affected the content of chlorophyll somewhat more than the content of the amino acids or protein nitrogen. However, spraying the plants with kinetin solution was able to retain 20–30% of the chlorophyll and protein. During senescence, the chlorophyll appears to be less stable in the N-deficient leaves than in the controls, while the protein is somewhat more stable than in the controls. Also, when the detached leaves from N-deficient plants senesced in white light or in darkness, kinetin delayed their senescence almost as effectively as that of control leaves. Most strikingly, the stomata of N-deficient leaves after detachment and floating on water were largely closed in light, just as in senescence, but could be partially induced to open by kinetin treatment. Since stomatal closure has earlier been shown to cause senescence, the characteristic syndrome of foliar nitrogen deficiency is concluded to be partly that of senescence.     

The role of protein synthesis in the senescence of leaves: I. The formation of protease

The senescence of oat leaves has been studied by following the loss of chlorophyll and protein and the increase of α-amino nitrogen, after detachment and darkening. Protein synthesis and the amounts of proteolytic enzymes in the leaves have been determined directly. The process of senescence is shown to be a sequential one in which protein synthesis,most probably the formation of a proteolytic enzyme with l-serine in its active center, is of prime importance. The evidence is as follows. Firstly, l-serine specifically enhances senescence, especially in presence of kinetin. Secondly, cycloheximide, which inhibits protein synthesis in other systems, delays senescence and prevents the serine enhancement. Although requiring higher concentrations, cycloheximide can be as effective as kinetin in inhibiting senescence. It is shown directly that cycloheximide prevents protein synthesis in oat leaves under the same conditions as when it prevents senescence. Thirdly, leaves have been shown to contain two proteinases, with pH optima at 3 and 7.5, whose activity increases during senescence, even though the total leaf protein is decreasing. The amounts of both these enzymes present after 3 days are clearly increased by serine, and are greatly decreased by cycloheximide or by kinetin. The role of kinetin in delaying senescence thus may rest on its ability to suppress protease formation.     

The interaction of hormonal and environmental factors in leaf senescence

The work concerns the senescence of isolated young leaves of oats (Avena sativa) floated on water or solutions. Senescence is rapid in darkness but slow in white light; the effect of light is not due to photosynthesis, but is paralleled by stomatal opening. Closure of the stomata by osmotic or chemical means makes senescence in light proceed as fast as in darkness, while opening the stomata in darkness by cytokinins, fusicoccin,etc., delays senescence to rates typical of light. The osmotic closure in light is mediated by abscisic acid, and since this also accumulates in darkness it appears as a major factor controlling senescence. Efflux of ions into the solution; indicating increased permeability, occurs almost in parallel with senescence. Senescence in light is accelerated by 1-aminocyclopropane-l-carboxylic acid (ACC) and inhibited by cobalt, silver or aminoethoxyvinyl glycine (AVG) which interfere with ethylene production or action; however, ethylene’s role is unclear because some reagents, including kinetin, that delay senescence, actually increase ethylene production. At the endogenous level, therefore, ethylene may not be a limiting factor. Finally, a new ethylene-generating system is described in which the dehydrogenation of linoleic acid is coupled through manganese to the oxidation of ACC; it is probably activein vivo.     

Recovery of loss in chlorophyll and 2,6-dichlorophenol indophenol Hill reaction of isolated chloroplasts during dark induced aging of intact maize seedlings

Loss in the content of pigments and decline in the efficiency of thylakoid membranes to reduce 2,6-dichlorophenol indophenol (DCPIP) have been investigated during dark induced senescence of attached leaves of maize seedlings. The chlorophyll degradation during senescence is differentially inhibited by indoleacetic acid (IAA), gibberellic acid (GA) and kinetin. IAA and GA behave as mild senescence inhibitors in comparison to kinetin. However, in comparison to light, kinetin is relatively less efficient in counteracting senescence. Dark-induced loss in chlorophyll content is fully recovered by light when the dark incubation period is relatively short. The pattern of light recovery of loss in photoelectron transport during dark-aging is similar to the recovery kinetics of chlorophyll. Continuous kinetin treatment of dark-incubated seedlings inhibits the chlorophyll degradation but with decreased duration of kinetin treatment, the efficiency of the hormone to inhibit chlorophyll loss is reduced. The kinetin-induced inhibition of pigment loss is small in comparison with the effect of light.     

Metabolism of Oat Leaves during Senescence: VI. CHANGES IN ATP LEVELS

The ATP content of 7-day-old Avena sativa leaves during senescence in dark and in light, and after treatment with cytokinins and other reagents, has been determined by the luciferin-luciferase method. Special care was taken to avoid decomposition of the ATP, and a detailed procedure is presented for ATP analysis at the picomole level. Preliminary experiments with several inhibitors of photophosphorylation suggest, though not conclusively, that the delaying effect of light on senescence is mediated by photophosphorylation. The ATP values of the leaves senescing in darkness are found to increase in parallel with the large increase in respiratory rate, and kinetin prevents this increase just as completely as it prevents the respiratory rise. It is concluded that the respiratory increase in senescence cannot be simply due to uncoupling. In light the ATP level also rises, though more slowly, and again kinetin prevents this rise. l-Serine, which promotes dark senescence, does not significantly modify the dark ATP level, but both arginine and kinetin, which antagonize the action of serine on senescence, greatly lower the ATP level below that on serine alone. Cycloheximide has a similar effect, and the combination of cycloheximide and kinetin lowers the ATP level drastically. Fusicoccin, which opens stomata in the dark, correspondingly maintains the ATP at a low level. Thus, in general, a low level of ATP is associated with the prevention of dark senescence, i.e. probably with ATP utilization, and the ATP level at any time may thus be determined more by the rate of utilization than by the efficiency of respiratory coupling.     

Relation between Respiration and Senescence in Oat Leaves

The respiration of excised oat (Avena sativa cv Victory) leaves and their sensitivity to inhibitors was followed during senescence under varied conditions. The respiration rate, which in controls reaches its peak on the third day in darkness, is lowered at the time of fastest loss of chlorophyll (as reported earlier) by seven unrelated reagents that all delay dark senescence. When senescence is delayed by white light or by cytokinins, the respiratory rise is correspondingly delayed. Kinetin and l-serine, which act as antagonists on senescence, also act as antagonists on the respiratory rate. However, an exception to this close correspondence between senescence and the respiratory rise is offered by the lower aliphatic alcohols, which delay dark senescence and yet accelerate the onset of the respiratory rise.     

Senescence in oat leaves: Changes in translatable mRNAs

Changes in translatable mRNA populations during the senescence of oat (Avena sativa L. cv. Victory) leaves were examined by analyzing the in vitro translation products of isolated RNA. Total RNA was isolated from oat leaves of 7-day-old seedlings, and also after these leaves were aged for different lengths of time under various conditions. Polypeptides from in vitro translations were separated by two-dimensional gel electrophoresis to estimate any changes in translatable mRNA populations associated with senescence. Corresponding leaf samples were monitored for loss of chlorophyll as a measure of the extent of senescence. The aging of excised leaves in the light for 4 days resulted in the disappearance or substantial quantitative decrease of a number of mRNA species, while only five new translatable mRNA species were produced. Three of these mRNAs were unique to aging of leaves under light. Two of these mRNA species were also produced during the early stages of senescence in attached leaves of seedlings grown under light. The translatable mRNA populations of leaves aged for 4 days either on intact seedlings or detached and kept in the light in the presence of kinetin were very similar. Aging of excised leaves in the dark on water for 24 h resulted in very extensive changes in translatable mRNA populations. Over thirty polypeptides disappeared or were substantially reduced in quantity, while about an equal number appeared de novo or were substantially increased in quantity. Aging of these leaves for an additional 24 or 48 h resulted in only a few additional changes in translatable mRNAs. The presence of kinetin during aging of excised leaves in the dark inhibited few of the numerous changes in mRNAs that occured during the first 24 h, but did inhibit most of the changes that occured after 48 or 72 h of aging in the dark. When leaves were first aged in the dark and then returned to light, most of the initial changes in translatable mRNAs expression were reversed. Such changes in mRNAs thus appear to be light-regulated and not necessarily associated with senescence.     

Interaction of polyamines and light on biochemical processes involved in leaf senescence

Abstract The rapid senescence of detached oat leaves in darkness is first manifested by a sharp rise in RNase activity (about 50% within 1 h), then by a rise in protease activity (indicated by an increase in non-protein α-amino nitrogen within 6 h) and ultimately by chlorophyll degradation (beginning after 18 h). These degradative changes are delayed or prevented by low concentrations (1–10 mM) of the naturally-occurring polyamines cadaverine, putrescine, spermidine and spermine. The tetraamine spermine is generally more active than the triamine spermidine, which is in turn more active than the diamines putrescine and cadaverine. All the polyamines are more active than kinetin or cycloheximide. As little as 10 min of exposure to 1 mM spermine, especially at the beginning of the dark period, produces a marked retardation of chlorophyll degradation over a 48 h period, and 60 min of exposure saturates the effect. In the light, all polyamines promote, rather than retard, the disappearance of chlorophyll but, as in the dark, they inhibit the rise in RNase and non-protein α-amino nitrogen. The photobleaching of chlorophyll in the presence of polyamines is proportional to the duration of exposure to high irradiance (16.5 Wm^?2) fluorescent light. Such light is more effective toward the end of the 48 h post-excision test period than at the beginning. Calcium ion (1–10 mM) supplied together with the polyamines diminishes their action in dark and light, indicating probable involvement of an initial ionic attachment mechanism. The loss of chlorophyll from the leaves of four species of dicotyledonous plants (pea, bean, rape, tobacco) in the darkness is similarly retarded by 1–10 mM polyamines. In rape, the most rapidly senescing species, 1 mM spermine almost completely arrests chlorophyll degradation over a 96 h period. It is suggested that polyamine metabolism in plants may be related to normal physiological control mechanisms as in microorganisms and animals, and that polyamines could find use as anti-senescence agents for plants.     

Possible mechanisms of light-induced chlorophyll degradation in senescing leaves of Hydrilla verticillata

Light treatment markedly accelerated the chlorophyll loss in senescing leaves of Hydrilla verticillata [(L.f.) Royle] as compared to dark treatment, whereas such acceleration could not be observed in senescing spinach (Spinacia oleracea L.) leaves. The light-induced cholorophyll loss in Hydrilla was retarded slightly by chloramphenicol and markedly by cycloheximide. Catalase (EC 1.11.1.6) activity did not change appreciably in Hydrilla leaves either in light or in darkness, while in spinach it declined markedly in the dark, and light retarded such decline. Peroxidase activity in Hydrilla showed faster increase in light than in darkness, while in spinach it increased only in light during senescence. The activity of phenol(pyrogallol)-specific peroxidase increased markedly in light, and that of ascorbate-specific peroxidase decreased slightly both in light and darkness during senescence of Hydrilla leaves. This rise in phenolspecific peroxidase activity was prevented by cycloheximide treatment. Pretreatment of Hydrilla leaves with monophenol (2,4-dichlorophenol) and o-diphenol (hydroquinone) accelerated and retarded, respectively, the light-induced cholorophyll loss. Pretreatment of Hydrilla leaves with H₂O₂ augmented the chlorophyll loss more markedly in light than in darkness. The endogenous level of H₂O₂ increased more in light than in dark during senescence of Hydrilla leaves. Treatment of Hydrilla leaves with 3-(3.4-dichlorophenyl)-l,l-dimethylurea. a photosystem II inhibitor, prevented both light-induced rise in H₂O: level and chlorophyll loss, but it was without effect in the dark. Retardation of light-induced chlorophyll loss occurred during senescence of Hydrilla leaves when light was given in different photoperiods in a 24-h daily cycle for 6 days instead of as continuous irradiance. There was a negative correlation between the length of the photoperiod and the extent of cholorophyll loss.     

Phytochrome and hormonal control of expansion and greening of etiolated wheat leaves

Summary Unrolling of etiolated wheat leaf segments is stimulated by short periods of exposure to red light. Both gibberellic acid and kinetin will stimulate unrolling in the dark, whereas abscisic acid (ABA) inhibits the unrolling response to these two hormones and to red light. Exposure to 5 minutes of red light leads to a rapid increase in endogenous gibberellin levels in etiolated wheat leaves, and this increase is followed by a rapid decline. Pre-treatment with ABA inhibits the increase in gibberellin levels in response to red light, but the ihibitory effect of ABA on unrolling cannot be ascribed only to its effect on gibberellin levels. Pre-treatment with red light reduces the lag-phase in chlorophyll development when wheat leaf segments are subsequently exposed to white light; the effect of red light may be replaced by pre-treatment with kinetin, but gibberellic acid is relatively ineffective in this respect.     

Endogenous gibberellin and abscisic Acid content as related to senescence of detached lettuce leaves

Levels of gibberillins (GAs) and of abscisic acid (ABA) in attached leaves of romaine lettuce (Lactuca sativa L.) declined as the leaf became older. The time course of changes in hormone levels, determined in detached lettuce leaves kept in darkness, revealed that a sharp decline in GAs accompanied by a moderate rise in ABA occurred before the onset of chlorophyll degradation. As senescence advanced, no GAs could be detected and a considerable rise of ABA was observed. A similar sequence of hormonal modifications, but more pronounced, was observed in the course of accelerated senescence induced by either Ethephon or water stress. When kinetin or GA₃ was applied to detached leaves, the loss of chlorophyll and the rise in ABA were reduced. Bound GAs were detected in senescent leaves. They were not found in the kinetin-treated leaves, which contained a relatively high level of free GAs. The results suggest that senescence in detached romaine lettuce leaves is connected with a depletion of free GAs and cytokinins, which is thereafter followed by a great surge in ABA.     

Metabolism of Oat Leaves during Senescence : VIII. The Role of L-Serine in Modifying Senescence

The mechanism whereby l-serine specifically promotes the dark senescence of detached oat (Avena) leaves has been examined. The fact that this promotion is strong in darkness but very weak in white light has been explained, at least in part, by the finding that added serine is partly converted to reducing sugars in light. Labeled serine gives rise to ¹⁴C-sugars and ¹⁴CO₂. In the absence of CO₂, serine does cause chlorophyll loss in light and undergoes a decreased conversion to sugar.     

Peroxidase and Senescence

Kinetin and a, á-dipyridyl prevented the rapid decreaseof chlorophyll content in detached oat leaves senescing in thedark. In the light, detachment caused a 27–40% rise in peroxidaseactivity and kinetin enhanced the enzyme in the segments byabout 80%. Darkness prevented any detachment-induced rise ofthe activity and decreased the stimulating action of kinetinand mechanical injury. The effect of dipyridyl on peroxidaseactivity in the dark was similar to that of kinetin. Kinetin enhanced the same distinctive isoperoxidases under lightand dark conditions. Neither horseradish peroxidase nor that extracted from oat leavesshowed any ability to hydroxylate free proline in vitro. A systemwhich supposedly led to peroxidase-catalysed proline hydroxylationyielded small amounts of hydroxyproline in the absence of theenzyme. Staining with Fast Blue BB salt in the presence of IAA as asubstrate after electrophoresis indicated that all detectedoat isoperoxidases had an IAA oxidase activity visually parallelingtheir peroxidase activity. Crude extracts contained IAA oxidaseinhibitors that could be partially or fully removed by dialysis. The possible significance of the rise in peroxidase activityduring senescence is discussed.     

The dependence of stomatal closure on protein synthesis

Seven different inhibitors of the synthesis of protein and RNA, all of which are found to delay the senescence of detached oat leaves in darkness, also cause the opening of the stomata in the dark. The concentration ranges for activity on the two processes agree closely. Four other compounds of similar effects on RNA and protein synthesis, but which are inactive on senescence, correspondingly fail to open the stomata. This not only strengthens the relationship between stomatal closure and senescence, but—more important—provides strong evidence that continued protein synthesis is necessary to keep foliar stomata closed.